At present, the primary objectives of forest-tree engineering and molecular breeding are to improve wood quality and yield. The global demand for wood products is growing at around 1.7% annually, and this increase in wood consumption is occurring despite the fact that the maximum sustainable rate of harvesting from the worlds forests has already been reached or exceeded. Therefore, there is a need for increases in plantation wood production worldwide. Forestry plantations may also have advantages as a carbon sequestration crop in response to increasing atmospheric CO2. Similarly, increased production of biomass from non-woody plants is desirable, for instance in order to meet the demand for raw material for energy production. Modification of specific processes during cell development in higher species is therefore of great commercial interest, not only when it comes to improving the properties of trees, but also other plants.
Plant growth by means of apical meristems results in the development of sets of primary tissues and in lengthening of the stem and roots. In addition to this primary growth, tree species undergo secondary growth and produce the secondary tissue “wood” from the cambium. The secondary growth increases the girth of stems and roots.
Sterky et al. 1998 (Proc. Natl. Acad. Sci. USA, 1998 (95), 13330-13335) have published the results of a large-scale gene discovery program in two poplar species, comprising 5,629 expressed sequence tags (ESTs) from the wood forming tissues of Populus tremula L.×tremuloides Michx. and Populus trichocarpa ‘Trichobel.’ These ESTs represented a total of 3,719 unique transcripts for the two cDNA libraries and putative functions could be assigned to 2,245 of these transcripts. The authors state that the EST data presented will be valuable in identifying genes involved in the formation of secondary xylem and phloem in plants, but fail to give clear directions as to how the identification could be performed. The Sterky et al. 1998 paper also revealed the existence of a very large number of ESTs with unknown or uncertain functions.
In the prior art (e.g. Sterky et al. 1998) libraries were constructed from stem tissue isolated from actively growing trees. A cambial region library was prepared from a mix of tissues, including the developing xylem, the meristematic cambial zone, and developing and mature phloem of P. Tremula×tremuloides Michx. These cambial tissues were obtained by peeling the bark and scraping both exposed surfaces with a scalpel. A developing-xylem library was prepared from Populus trichocarpa Tricobel. These tissues were obtained by peeling the bark and scraping the exposed xylem side. Using such methods it is only possible to build three different libraries representing the whole cambial region, the developing-xylem and the phloem region (made from scraping the exposed bark). The prior art compared the expression of genes in the cambial-region with the genes expressed in the developing xylem tissue. The experiment only allowed a crude comparison due to the limits imposed by the tissue preparation protocol. The tissue used for the developing xylem library would contain tissues from expanding xylem cells through to late xylem development.
One problem remaining is how to identify the potentially most important genes and to relate these to specific developmental stages and final properties of the cell. Another problem is how to identify hitherto unknown genes, related to specific cell types and/or functions in the plant. Finally, a particular problem is how to find the specific genes involved in cell division, cell expansion, cell wall synthesis, apoptosis and programmed cell death and other important processes involved in determining tree growth and wood properties.
Hertzberg et al. 2001 (Proc. Natl. Acad. Sci. USA, 2001 (98), 14372-14737), and Schrader et al. 2005 (Plant Cell, (16), 2278-2292) have used transcript profiling to reveal a transcriptional hierarchy for thousands of genes during xylem development as well as providing expression data that can facilitate further elucidation of many genes with unknown function (White et al. 1999 (Science 1999 (286) 2187-2184); Aharoni et al. 2000 (Plant Cell 2000 (12) 647-662). This is however technically demanding in woody plants such as trees. Hertzberg et al. and Schrader et al. have studied the developing secondary xylem of poplar, which is highly organized with easily recognized and distinct boundaries between the different developmental stages. Wood formation is initiated in the vascular cambium. Cambial derivatives develop into xylem cells through the processes of division, expansion, secondary wall formation, lignification and, finally, programmed cell death. The large physical size of the vascular meristem in trees offers a unique possibility to obtain samples from defined developmental stages by tangential cryo sectioning (Uggla et al. 1996 Proc. Natl. Acad. Sci. USA, 1996 (93), 9282-9286). To determine the steady state mRNA levels at specific stages during the ontogeny of wood formation in Populus tremula×tremuloides (hybrid aspen) 30 μm thick sections through the wood development region were sampled and subsequently analyzed using several spotted cDNA-microarray (Schena et al. 1995 Science 1995 (270) 467-470) consisting of up to 20.000 unique ESTs from hybrid aspen.
Although it is obvious that results from EST programs, genome sequencing and expression studies using DNA array technologies can verify where and when a gene is expressed it is rarely possible to clarify the biological and/or technical function of a gene only from these types of analytical tools. In order to analyze and verify the gene function a functional characterization must be performed, e.g. by gene inactivation and/or gene over-expression. However, in order to be able to identify genes with interesting and most often unexpected commercial features, there is a need for novel analytical platforms evaluating candidate genes based on multiple criteria.